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Creation of proteinaceous insecticide using the technology of directed evolution against Bacillusthuringiensis insecticidal toxins

Research Project

Project/Area Number 18310053
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Environmental technology/Environmental materials
Research InstitutionTokyo University of Agriculture and Technology

Principal Investigator

RYOICHI Sato  Tokyo University of Agriculture and Technology, Institute of Symbiotic Science and Technology, Associate Professor (30235428)

Project Period (FY) 2006 – 2007
Project Status Completed (Fiscal Year 2007)
Budget Amount *help
¥17,630,000 (Direct Cost: ¥15,800,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2007: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2006: ¥9,700,000 (Direct Cost: ¥9,700,000)
Keywordsbiotechnology / protein / microorganism / Bacillus thruingiensis / insecticide
Research Abstract

Bacillus thuringiensis produces Cry toxins, insecticidal proteins which are induced production at the time of the sporulation. It is theoretically possible to make activity improved toxin by increasing affinity of the toxin to the receptor altering amino-acid sequences especially of the receptor-binding region of the toxin. In this research, we used a phage display system, which was developed in our laboratory, and tried to achieve the directed evolution of Cry toxins to increase the affinity to the receptor. To clarify the receptor binding region, first we constructed 13 cysteine-replaced mutant toxin DNA by site-directed mutagenesis. After production of mutant toxins, we utilized the thiol of the cysteine to be bound with some molecules as inhibitors of receptor-binding, and analyzed these mutants whether inhibition occurred or not. From these experiments we concluded that face made by loop 2, loop 3 and loop alpha 8 is important as a receptor binding site. Next we constructed 11 phage libraries displaying mutant Cry toxin with changed amino acids at loop 2, loop 3 or loop alpha 8. Selection of mutant Cry toxin by bio-panning or magnetic beads system was conducted. From some libraries, concentration of some mutant toxin displaying phages was observed. Although we are now confirming the actual insecticidal activity and improvement of binding affinity to the receptor of each mutant toxin, we are expecting that those mutants have improved insecticidal activities.

Report

(3 results)
  • 2007 Annual Research Report   Final Research Report Summary
  • 2006 Annual Research Report
  • Research Products

    (30 results)

All 2008 2007 2006

All Journal Article (11 results) (of which Peer Reviewed: 7 results) Presentation (19 results)

  • [Journal Article] Selective inhibition of binding of Bacillus thuringiensis CrylAb toxin to cadherin-like and aminopeptidase proteins in brush border membranes and dissociated epithelial cells from Bombyx mori.2008

    • Author(s)
      M. S. Ibiza-Palacios
    • Journal Title

      Biochem J. 409

      Pages: 215-221

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Histochemical analysis of Bacillus thuringiensis CrylA toxin binding to midgut epithelial cells of Bombyx mori2008

    • Author(s)
      D. M. Hossain
    • Journal Title

      Pesticide Biochemistry and Physiology 87

      Pages: 30-38

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Selective inhibition of binding of Bacillus thuringiensis CrylAb toxin to cadherin-like and aminopeptidase proteins in brush border membranes and dissociated epithelial cells from Bombyx mori.2008

    • Author(s)
      M. S., Ibiza-Palacios
    • Journal Title

      Biochem J. 409

      Pages: 215-221

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Selective inhibition of binding of Bacillus thuringiensis CrylAb toxin to cadher in-like and aminopeptidase proteins in brush border membranes and dissociated epithelial cells from Bombyx mori.2008

    • Author(s)
      M. S. Ibiza-Palacios
    • Journal Title

      Biochem J. 409

      Pages: 215-221

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Binding of Bacillus thuringiensis CrylA toxins to brush border membrane vesicles of midgut from CrylAc susceptible and resistant Plutella2007

    • Author(s)
      M. Higuchi
    • Journal Title

      Comp Biochem Physiol Part B 147

      Pages: 716-724

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.2007

    • Author(s)
      H. Ishikawa
    • Journal Title

      Molecular Biotechnology 36

      Pages: 90-101

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Annual Research Report 2007 Final Research Report Summary
    • Peer Reviewed
  • [Journal Article] Histochemical analysis of Bacillus thurin-giensis CrylA toxin binding to midgut epithelial cells of Bombyx mari2007

    • Author(s)
      D. M., Hossain
    • Journal Title

      Pesticide Biochemistry and Physiology 87

      Pages: 30-38

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Binding of Bacillus thuringiensis CrylA toxins to brush border membrane vesicles of midgut from CrylAc susceptible and resistant Plutella xylostella.2007

    • Author(s)
      M., Higuchi
    • Journal Title

      Comp Biochem Physiol Part B 147

      Pages: 716-724

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.2007

    • Author(s)
      H., Ishikawa
    • Journal Title

      Molecular Biotechnology 36

      Pages: 90-101

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Journal Article] Histochemical analysis of Bacillus thuringiensis CrylA toxin binding to midgut epithelial cells of Bombyx mori2007

    • Author(s)
      Delwar M. Hossain
    • Journal Title

      Pesticide Biochemistry and Physiology 87

      Pages: 30-38

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Binding of Bacillus thuringiensis CrylA toxins to brush border membrane vesicles of midgut from CrylAc susceptible and resistant Plutella xylostella.2007

    • Author(s)
      M. Higuchi
    • Journal Title

      Comp Biochem Physiol Part B 147

      Pages: 716-724

    • Related Report
      2007 Annual Research Report
    • Peer Reviewed
  • [Presentation] Selection of high affinity Cry toxin mutant to BtR175 using phage display.2008

    • Author(s)
      T. Kotani
    • Organizer
      Asia-Pacific Congress of Sericulture and Insect Biotechnology
    • Place of Presentation
      名古屋大学
    • Year and Date
      2008-03-22
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Annual Research Report 2007 Final Research Report Summary
  • [Presentation] Binding region of insecticidal protein CrylAa from Bacillus thuringiensis for receptor in B. mori.2008

    • Author(s)
      F. Obata
    • Organizer
      Asia-Pacific Congress of Sericulture and Insect Biotechnology
    • Place of Presentation
      名古屋大学
    • Year and Date
      2008-03-22
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Selection of high affinity Cry toxin mutant to BtR175 using phage display2008

    • Author(s)
      T., Kotani
    • Organizer
      Asia-Pacific Congress of Sericulture and Insect Biotechnology
    • Place of Presentation
      Nagoya, Japan
    • Year and Date
      2008-03-22
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Binding region of insecticidal protein CrylAa from Bacillus thuringiensis for a receptor in B. morl2008

    • Author(s)
      F., Obata
    • Organizer
      Asia-Pacific Congress of Sericulture and Insect Biotechnology
    • Place of Presentation
      Nagoya, Japan
    • Year and Date
      2008-03-22
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Binding region of insecticidal protein CrylAa from Bacillus thuringiensis for a receptor in B. mori.2008

    • Author(s)
      F. Obata
    • Organizer
      Asia-Pacific Congress of Sericulture and Insect Biotechnology
    • Place of Presentation
      名古屋大学
    • Year and Date
      2008-03-22
    • Related Report
      2007 Annual Research Report
  • [Presentation] ファージディスプレイ法を用いたBtR175高親和性を有したCry毒素変異体の選抜2007

    • Author(s)
      森本智夏
    • Organizer
      第30回日本分子生物学会
    • Place of Presentation
      パシフィコ横浜
    • Year and Date
      2007-12-14
    • Related Report
      2007 Annual Research Report
  • [Presentation] 受容体に高親和性を有したCry毒素変異体の進化分子工学的手法による選抜2007

    • Author(s)
      原島裕子
    • Organizer
      第69回昆虫病理研究会講演会
    • Place of Presentation
      文部科学省研究交流センター
    • Year and Date
      2007-12-07
    • Related Report
      2007 Annual Research Report
  • [Presentation] ファージディスプレイ法を用いたBtR175高親和性を有したCry毒素変異的の選抜2007

    • Author(s)
      小谷拓也
    • Organizer
      第13回BT研究会
    • Place of Presentation
      新潟県西蒲原郡弥彦村やひこ荘
    • Year and Date
      2007-09-27
    • Related Report
      2007 Annual Research Report
  • [Presentation] Development of the proteinaceous inseciticide from a soil bacterium (Bacillus thuringiensis) using Phage display2007

    • Author(s)
      Delwar M Hossain
    • Organizer
      第13回BT研究会
    • Place of Presentation
      新潟県西蒲原郡弥彦村やひこ荘
    • Year and Date
      2007-09-27
    • Related Report
      2007 Annual Research Report
  • [Presentation] An approach to the directed evolution of the insecticidal protein from Bacillus thuringiensis.2006

    • Author(s)
      Y. Hoshino
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control,
    • Place of Presentation
      Wuhan, China.
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Bacillus thuringiensis Cry toxin' s domain III, Galactose-binding Domain-like binds specifically to various proteins.2006

    • Author(s)
      M. Kitami
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control,
    • Place of Presentation
      Wuhan, China.
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Antibody blocking of putative receptors inhibits the binding of CrylAb in Bombyx mori.2006

    • Author(s)
      M. S. Ibiza-Palacios
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control,
    • Place of Presentation
      Wuhan, China.
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Lack of irreversible binding as a novel mechanism of resistance to Bacillus thuringiensis Cry1Ab toxin.2006

    • Author(s)
      M. S. Ibiza-Palacios
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control,
    • Place of Presentation
      Wuhan, China.
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] An approach to the directed evolution of the insecticidal protein from Bacillus thuringiensis2006

    • Author(s)
      Y., Hoshino
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control
    • Place of Presentation
      Wuhan, China
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Bacillus thuringiensis Cry toxin's domain III, Galactose-binding Domain-like binds specifically to various proteins2006

    • Author(s)
      M., Kitami
    • Organizer
      9th colloquium on invertebrate pathology and microbial control
    • Place of Presentation
      Wuhan, China
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Antibody blocking of putative receptors inhibits the binding of CrylAb in Bombyx mari2006

    • Author(s)
      M. S., Ibiza-Palacios
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control
    • Place of Presentation
      Wuhan, China
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] Lack of irreversible binding as a novel mechanism of resistance to Bacillus thuringiensis CrylAb toxin2006

    • Author(s)
      M. S., Ibiza-Palacios
    • Organizer
      9th international colloquium on invertebrate pathology and microbial control
    • Place of Presentation
      Wuhan, China
    • Year and Date
      2006-08-28
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] A system for the directed evolution of the insecticidal protein from Bacillus thuringiensis.2006

    • Author(s)
      R. Sato
    • Organizer
      20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
    • Place of Presentation
      Kyoto, Japan.
    • Year and Date
      2006-06-20
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2007 Final Research Report Summary
  • [Presentation] A system for the directed evolution of the insec-ticidal protein from Bacillus thuringiensis.2006

    • Author(s)
      R., Sato
    • Organizer
      20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
    • Place of Presentation
      Kyoto, Japan
    • Year and Date
      2006-06-20
    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2007 Final Research Report Summary

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Published: 2006-04-01   Modified: 2016-04-21  

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